Viral infection is not only extremely harmful to human health, but also severely endangers the survival and breeding of various animals, and thus becoming an important research task in medical and relevant fields currently. Investigation and invention of a medicine for treating viral infection has a significant application potential. Therefore, once succeeds, it will generate a huge social and economic benefit.
Influenza (shortened as “flu”) is the most rampant infectious disease in the world. This acute infectious disease of upper respiratory tract has some features, such as a strong infectivity, a rapid spreading ability, a short latent period, and a high morbidity. And influenza is a viral infectious disease commonly suffered in human, avian and livestock. Pandemic influenza occurred several times in the past: the pandemic influenza of 1918 in Spain, which claimed 50 million lives; the pandemic influenza of 1957, which led to 15 hundred millions infected globally. The morbidity, mortality as well as social and personal economic damage caused by influenza are still ranked as No. 1 in all infectious diseases, which brings a huge calamity to human. Influenza is a frequently occurred disease in China. In fact, several pandemics of influenza since 1957 originated from China (Yuanji Guo, Xiaowen Cheng, 1997). Therefore, prophylaxis and treatment of influenza is well appreciated in China.
Influenza virus is the pathogen causing influenza, which belongs to Orthomyxoviridae family, and is a negative-strand RNA virus varying frequently. Highly Pathogenic Avian Influenza Virus (HPAIV), which has drawn a lot of attention for these years, is a highly contact acute infectious disease caused by influenza A virus. It causes many lethal pathologic changes to both domestic fowls and wild birds, including the pathologic change of respiratory systems to systematic septemia. Worldwide scale pandemics of HPAIV have occurred many times. Even worse, the unprecedented infection of avian influenza virus subtype H5N1 in human has occurred several times for the past a few years, and resulted in death of patients (Kamps et al, 2006). The possibility of highly pathogenic avian influenza virus to spread among human by crossing the barrier between species and to cause a new round worldwide pandemic makes all countries attach importance to the prophylaxis and treatment of the highly pathogenic avian influenza. HPAIV has been ranked as a category A infectious disease by Office International Des Epizooties (OIE), and listed as one of the animal infectious diseases in International Biological Weapon Treaty. HPAIV was also ranked as a category A monitored infectious disease by Department of Agriculture in China.
Influenza virus is essentially consisted of envelope and nucleocapsid, wherein the envelope is composed of spike, bilayer lipid membrane and matrix protein (M). The spike is divided into two categories: one is rod-shaped and consisted of trimers of hemagglutinin (HA) protein; the other is mushroom-shaped and consisted of tetramers of neuraminidase (NA). The bilayer lipid membrane is obtained from host cells membrane while the virion is budding. The matrix protein is closely lined up at the inner side of the envelope, and is a structural protein maintaining the viral shape. The nucleocapsid, coated by the matrix protein, is consisted of 8 RNA-nucleoprotein complexes (RNP) arranged in a helix shape. Each RNP is consisted of monomer negative-strand RNA coated with nucleoprotein (NP). The 8 negative-strand RNAs in influenza virus genome thus constitute 8 RNPs. According to the different antigenicity between nucleoprotein NP and matrix protein M, influenza virus is divided into 3 types, i.e. A, B and C. The 3 types of influenza virus do not share any common antigen, but all infect human. Among them, influenza A virus has the biggest pandemic scale. Each type of influenza virus is further divided into many subtypes according to the different antigenicity of hemagglutinin (HA; shortly as “H”) and neuraminidase (NA; shortly as “N”) at the viral surface. There has been found 16 subtypes of H, 9 subtypes of N in influenza A virus. Subtype H and N may constitute various combinations, such as H1N1, H5N1, H3N2, and H9N2, etc.
Since 1918, it has been witnessed several worldwide pandemic and some epidemic of influenza caused by influenza virus. Influenza virus is very rampant, since it constantly varies. The reasons causing the variation mainly include: 1) influenza virus genome is consisted of negative-strand RNA and RNA replicase lacks of a correction function during the genomic replication, therefore it is very easy for the viral genome to be varied during the replication in host cells, which leads to the variation of the amino acids encoded by it and thereby leads to an antigen shift; 2) influenza virus genome is consisted of 8 of segmented RNAs, and the 8 segments can be randomly reassorted while one cell is infected with various influenza virion. Theoretically, 28 (256) of new viral strains with completely different antigenicity and pathogenicity may be formed through the gene reassortment, therefore an antigen shift will occur, which enables influenza virus to escape the monitor of the immune system, to replicate in a large scale in host cells, and to cause new pandemic of influenza. As a result, the antigen of influenza virus varies rapidly and the reassortment rate among different influenza viral strains is very high, which leads to a huge challenge to the prophylaxis and treatment of influenza virus.
Vaccination of influenza virus is the first strategy for preventing human influenza currently. However, influenza virus has many serotypes, so if the antigenicity between the vaccine strain and epidemic strain does not match each other, the vaccine will lose its function, and may not provide corresponding protection. On the other hand, since influenza virus varies frequently, the speed of vaccine development is far behind that of influenza virus variation. In fact, when a new epidemic strain appears, at least 6 months are required for producing its corresponding vaccine (Kamps et al, 2006), which makes the preparation of vaccines always in a passive position. In this case, neither a traditional inactivated vaccine nor a novel vaccine such as genetic engineering vaccine, a nucleic acid vaccine and the like can provide crossing protection for all types of influenza virus. Moreover, the protection period for influenza vaccines is very short, i.e. only half a year to a year, so these vaccines need to be injected every year, which is hard to be accepted by the patients. However, regarding vaccines for the highly pathogenic avian influenza virus, a policy for forbidding or not advocating the utilization of such vaccines is taken in many countries, so as to avoid frequently occurred variation of the viral antigens caused under a selective pressure of hosts. The use of such vaccines is also under disputation in academia (Kamps et al, 2006).
Due to the relatively poor effect of vaccines on prophylaxis of influenza, the investigation of pharmaceuticals for preventing and treating influenza virus has drawn a lot of attention. Till now, it has already gained some advances in development and research of the pharmaceuticals designed upon the molecular mechanism regarding viral infection and replication.
There are two classes of chemotherapeutic agents for influenza: (1) ion channel inhibitor, i.e. Amantadine and Rimantadine targeting the ion channel protein M2 of influenza virus, which prevent the replication of influenza virus by interfering the ion channel activity of influenza virus M2 protein (Skehel, 1992). Although such a chemotherapeutic agent results in certain therapeutic effect, a large amount of drug-resistant influenza virus strains have appeared (Jefferson et al, 2006), and such agent causes more severe toxic side effect. As a result, the specialists in WHO have suggested to stop using M2 ion channel inhibitors as anti-influenza medicines (Kamps et al; 2006). (2) neuraminidase inhibitor, i.e. an inhibitor targeting NA of influenza virus, such as 4-guanidino-2,4-deoxy-2,3-dehydro-N-acetylneuraminicacid (4-guanidino-Neu5Acen) and an analog thereof, which may effectively inhibit the release of virion at the host cell surface by inhibiting the activity of NA, thereby preventing other host cells from being infected with influenza virus. Such chemotherapeutic agents, with the trade names of Zanamivir and Oseltamivir, are used to treat influenza virus in USA and Australia, etc. Leneva et al. (2001) carried out an in vitro experiment using an influenza virus-sensitive cell line, i.e. MDCK, which showed that the 50% effect concentration (EC50) of Zanamivir is 8.5-14.0 μM for the 3 subtypes of virus i.e. H5N1, H6N1 and H9N2. Although such agent has less toxic side effect than ion channel inhibitor, the corresponding drug-resistant strains also appeared (Abed et al, 2006), especially in the patients infected with avian influenza virus H5N1 (de Jong, 2005). (3) artificially synthesized sialosyl oligosaccharide analog, such as analogs of sialosyl glucoside liposome, sialosyl glucoside multimer, and bi-valence sialosyl glucoside, etc, which competitively binds to HA, thereby prohibiting HA from binding to the surface receptors on the host cell membrane, interfering the viral adsorption, and thereby suppressing the viral function. Till now, such suppression has been confirmed in an in vitro experiment (Haolong Pang et al, 2004), but in vivo data has not been reported, and the corresponding commercial medicines are not available. (4) A variety of anti-influenza A virus single-component and compound preparations of Chinese medicines, mainly including exterior-releasing medicines and heat-clearing and detoxifying medicines, have been screened out (Weimin Zhang et al, 2001) as a supportive therapy and they are not the inhibitors specified for influenza virus. Additionally, administration of Chinese medicine is so inconvenient that it can be only accepted by a few people. Moreover, the effect of Chinese medicine is still disputable in the western world. As a result, the anti-influenza virus Chinese medicines are very difficult to be widely used in a global extent.
There has gained some advance in the development of anti-influenza medicines using biotechnology. For example, in laboratory research, an oligonucleotide sequence synthesized or an expression vector constructed using anti-sense DNA technique, anti-sense RNA technique or RNA interfering technique (siRNA) show different levels of anti-influenza virus activities (Gao et al, 2006). However their corresponding commercial medicines are still not available. Some interrelated research indicated that the variation of siRNA targeting sequences would also result in appearance of the virus-tolerant strains. For instance, currently, a mutated drug-resistant strain has been found in the studies of anti-poliomyelitis virus and human immunodeficiency virus I (HIV-1) (Das et al, 2004).
At present, it shows a good prospect in the development of the anti-virus medicine by which a 7-peptide repeat region (Heptad Repeat, HR), i.e. a conserved region on main membrane protein of enveloped virus, is used as a target, wherein its mechanism is shown as follows: the critical step of infecting host cells with enveloped virus is the step of fusion of the membranes between the virus and the host cell, wherein the membrane fusion process is mediated by the fusion protein on the virus envelope. The fusion proteins can be divided into 2 groups, i.e. type I and II, based on their structural features. The virus containing fusion protein I is very common, including orthomyxovirus, paramyxovirus, coronavirus, retrovirus and filovirus, etc. The structure of fusion protein I comprises two regions of so called “7-peptide repeat (HR) sequence”, wherein the one at the N-terminal of the fusion protein is designated as HR1, and the one at the C-terminal of the fusion protein is designated as HR2. These two sequences play important roles when fusion protein I exerts a membrane fusion activity. The locations and lengths of sequences HR1 and HR2 vary in different viruses. During the process of fusion, 3 of HR2s attach into the groove of a central trimer constituted with 3 of HR1s in an antiparrallel manner, thereby forming a stable 6-helix bundle or a structure called as a hairpin trimer. During the formation of such structure, the viral envelope and cell membrane may be drawn closer and then contacted with each other, which induces fusion of the membranes. The studies regarding HIV-1, SARS coronavirus (SARS-CoV), human respiratory syncytial virus (HRSV), and newcastle disease virus (NDV) show that the exogenously incorporated HR1 or HR2 polypeptides may inhibit infection of host cells with virus, wherein the mechanism thereof was presumed as follows: during the conformational variation of membrane proteins and the induction of membrane fusion, the exogenous polypeptides may competitively bind to the HR1 or HR2 on the virus fusion protein, thus interdicting the interaction between HR1 and HR2 of the virus fusion protein itself. Therefore, the hairpin trimer structure of the virus fusion protein could not be formed, which inhibits the fusion between viral envelope and cell membrane, thus preventing virus from entering into cells (Young et al, 1999; Wang et al, 2003; Greenberg et al, 2004). At the present, the polypeptide T-20 derived from the HR2 region on HIV-1 fusion protein gp41 was approved by the “express way” of Food and Medicine Administration (FDA) of USA, becoming the first anti-AIDS polypeptide medicine with excellent therapeutical effect for preventing HIV-1 from entering into cells. However, the artificial synthesis of such a polypeptide medicine is very expensive, and the treatment of HIV-1, a chronic infectious virus, requires a long term injection, so the injection of T-20 costs USD 20,000/year in USA, and USD 25,000/year in Europe. As a result, the application of T-20 is restricted by high cost. On the contrary, influenza virus causes transient but not long term chronic infection. In this case, if the prophylaxis and treatment by administration is timely performed before and after infection, it is possible to effectively control the progression of the disease, reduce infectivity, and decrease mortality of the infected subjects.
In view of the above, it is indicated that although there has gained some advance in the development and research of anti-influenza virus medicines, the tolerant strains for all commercialized chemical compound medicines appeared. Among the current anti-influenza medicines protected by patents, none of them is commercially available and is developed on the basis of biotechnology.